The current blood banking system has inherent risks and serious limitations. Blood typing errors, transmission of bacterial agents, and viral infections such as HIV-1 and hepatitis A, B, and AB pose life threatening dangers to transfusion patients. In addition, availability of donors, requirement for specific blood types, short shelf life of red blood cells, and need for refrigeration all limit the accessibility of a transfusion to a patient. Development of a stable blood substitute could eliminate the risks of the current blood banking system and increase the availability of transfusions to patients in most environments.
In addition, an oxygen carrying blood substitute can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, and can also support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution can be used in most situations where red blood cells or plasma expanders are currently utilized. An oxygen-transporting hemoglobin-based solution could also be used to temporarily augment oxygen delivery during or after pre-donation of autologous blood prior to the return of the autologous blood to the patient.
However, several obstacles must be overcome in the development of an optimal hemoglobin-based oxygen carrier, including: (1) inhibition of tetramer to dimer dissociation; (2) reduction of hemoglobin oxygen affinity; (3) inhibition of autooxidation; (4) inhibition of heme loss; and (5) increased stability of the apoglobin tertiary structures.
Thus far, most hemoglobin-based blood substitute designs have successfully focused on preventing tetramer dissociation and reducing oxygen affinity through chemical and genetic techniques (Winslow, R. M. (1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). Gawryl and coworkers (Gawryl, M., Clark, T., and Rausch, C., in Red Cell Substitutes: The Proceedings of the Second International Symposium of Red Blood Cell Substitutes (S. Sekiguchi, ed.) pp. 28-40, Tokyo:Kindai Shupann, 1991) chemically cross-linked bovine hemoglobin using glutaraldehyde to prevent tetramer dissociation. Besides being very abundant, bovine hemoglobin was chosen because its oxygen affinity is regulated by chloride ions, and, as a result, has a relatively high P.sub.50 value, which is the same inside or outside a red blood cell. Chatterjee et al. (Chatterjee, R., Welty, E. V., Walder, R. Y., Pruitt, S. L., Rogers, P. H., Arnone, A., and Walder, J. A. (1986) J. Biol. Chem. 261, 9929-9937) chemically crosslinked human hemoglobin using (3,5-dibromosalicyl)fumarate under conditions which also caused a two fold decrease in the oxygen affinity of the modified protein. A genetic approach is discussed by Hoffman et al., who used recombinant hemoglobin genes and an E. coli. expression system (Hoffman et al., WO 90/13645). They genetically linked the C-terminal residue of one alpha subunit to the N-terminus of the other alpha subunit using a flexible glycine residue, producing a single alpha.sub.1 -alpha.sub.2 subunit (Looker, D., et al. Methods in Enzymology 231, 364-374, 1994). The tandem alpha globin gene was then combined with a copy of the beta globin gene and placed under the control of a single promoter to form a hemoglobin operon. To decrease the oxygen affinity, they also incorporated the Presbyterian mutation into the beta subunits. Presbyterian mutation refers to the beta(G10)Asp.fwdarw.Lys substitution which causes a reduction in oxygen affinity in both hemoglobin subunits. The final protein was designated rHb1.1 and has a P.sub.50 value similar to that observed for intact red blood cells.
Inhibition of tetramer dissociation and alteration of oxygen affinity are only the first steps towards creating an optimal cell-free hemoglobin based blood substitute. The next step is to minimize the effects that may result from the instability of hemoglobin by increasing resistance to autooxidation, hemin loss, and apoglobin denaturation. Potentially toxic effects may result from the by products of autooxidation, free hemin, and insoluble apoglobins. These problems can include oxidative and peroxidative tissue damage and propagation of free radicals, (Vandegriff, K. D. (1995) in Blood Substitutes: Physiological Basis of Efficacy (Birkhauser, Boston) pp. 105-131). Moreover, the globin subunits that contain an oxidized iron or that have lost heme can no longer transport oxygen.
Hemoglobin is a tetrameric protein consisting of two alpha and two beta subunits and is the oxygen binding component in red blood cells. Each of the subunits is composed of a globin (protein portion) and a heme prosthetic group. Each of the globins fold into 8 alpha helices, labeled A through H, with the exception that alpha subunits lack five residues which correspond to the beta subunit D helix. The only nonhelical segments are the turns between helices. The positions of the amino acids are denoted by their position within a particular helix or their distance from the N-terminus. Hemoglobin subunits produced recombinantly by the methods described herein have an N-terminal methionine in place of the normally occurring N-terminal valine. The N terminus, regardless of identity is denoted as amino acid number 1.
In each globin, ligands bind to the 6.sup.th coordination site of iron in the heme prosthetic group, protoporphyrin IX. The heme group is secured to the globin by a covalent bond between the 5.sup.th coordination site of the heme iron and a proximal His(F8) residue. The ferrous or reduced state (Fe.sup.+2) binds oxygen and carbon monoxide. The ferric form, known as methemoglobin, binds water, azide, cyanide, or hydroxide anions. When the iron in the protoporphyrin IX is reduced, the prosthetic group is denoted heme. On the other hand, when the iron is in the oxidized state in the protoporphyrin IX, the complex is called hemin.
Hemoglobin binds some ligands cooperatively. Cooperativity allows efficient O.sub.2 uptake in the lungs where oxygen partial pressure is high and release in muscle capillaries where the partial pressure is much lower. Cooperative O.sub.2 binding to hemoglobin is a result of allosteric interactions between the alpha and beta subunits.
The alpha.sub.1 beta.sub.1 dimer is predominantly held together through strong hydrophobic interactions between the two subunits. Formation of the hemoglobin tetramer results from relatively weaker electrostatic interactions between two alpha.sub.1 beta.sub.1 dimers, resulting in a tetramer with two new subunit interfaces called alpha.sub.1 beta.sub.2 and alpha.sub.2 beta.sub.1.
The hemoglobin tetramer can exist in either the T (low oxygen affinity) or the R (high oxygen affinity), quaternary conformation. In the absence of oxygen, hemoglobin is held in the T state by a lattice of electrostatic interactions at the alpha.sub.1 beta.sub.2 and alpha.sub.2 beta.sub.1 interfaces. Interconversion between the T and R states is accomplished by rotating the alpha.sub.1 beta.sub.1 dimer 15.degree. with respect to the alpha.sub.2 beta.sub.2 dimer, or vice versa. The alpha.sub.1 beta.sub.1 and alpha.sub.2 beta.sub.2 interfaces are not affected by T to R interconversion, but formation of the R state requires disruption of a significant number of the electrostatic bonds in the T-state alpha.sub.1 beta.sub.2 and alpha.sub.2 beta.sub.1 interfaces.
Methemoglobin is formed by the oxidation of the heme iron from Fe.sup.+2 to Fe.sup.+3 (Winterbourn, C. C., and Carrell, R. W. J. Clin. Invest. 54, 678, 1977; Bunn, H. F., and Forget, B. G., Hemoglobin: Molecular, Genetic, and Clinical Aspects (W. B. Saunders Co.) Philadelphia, Pa., 1986). This methemoglobin is physiologically inactive since it does not bind oxygen. Moreover, hemin can readily dissociate from the methemoglobin molecule because the bond between the iron atom and His93 (F8) is considerably weakened upon oxidation of the iron. Due to the insolubility of free hemin and apoglobin at physiological pH and temperature, hemin dissociation is essentially irreversible.
The affinity of the globins for heme is regulated through a combination of covalent, hydrophobic, electrostatic, and steric effects between the globins and bound hemin. The covalent bond between the His(F8) residue and the fifth coordination site of iron is an important force securing heme to the ferrous globins. However, after autooxidation this bond is considerably weakened, resulting in a faster rate of hemin dissociation from methemoglobin than from ferrous hemoglobin. Hydrophobic interactions between the methyl and vinyl substituents of the tetrapyrrole ring and the apolar regions of the globin make an important contribution to the retention of heme. Hydrogen bonding between His64(E7) and coordinated water helps to anchor heme in the globin. Salt bridges between polar amino acid residues at the surface of the globin and the heme-6- and heme-7-propionates also inhibit hemin loss. The heme-7-propionate forms hydrogen bonds with Lys(E10) in alpha and beta globin. The heme-6-propionate forms a salt bridge with His45(CE3) in alpha globin (Bunn, H. F. and Forget, B. G. (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects, Chapter 16, pages 634-662, W. B. Saunders Company, Philadelphia, Pa.). The equivalent residue in the beta subunit, Ser44(CD3), is too far from the heme-6-propionate to form a similar interaction, and this lack of stabilization may contribute to the rapid rate of hemin loss from hemoglobin beta subunits.
The polypeptide chain between the C and E helices in alpha globins is 5 residues shorter than the equivalent region in beta globin, resulting in loss of helical secondary structure in this region of the protein (Kleinschmidt, T. & Squoros, J. Hoppe-Seyler's Z. Biol. Chem. 368, 579-615, 1987). Komiyama et al. (Komiyama, N., Shih, D., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 49-51) examined the functional significance of the D helix in beta globins and its loss from alpha globins. No decrease in cooperativity or marked increase in O.sub.2 affinity was observed. Komiyama et al. concluded that loss of the D-helix from alpha subunits was a functionally neutral mutation with respect to O.sub.2 binding and assembly into a cooperative tetramer. However, this left unresolved the origin of the strong selective pressure to preserve a D-helix in the beta subunits of vertebrate hemoglobins.
Isolated hemoglobin subunits are highly unstable and lose hemin more readily than myoglobin. The resultant apohemoglobins are highly unstable at physiological pH and temperature. Because of the instability of hemoglobin subunits, myoglobin has been used as a model system to understand the principles of globin folding and stability. Apomyoglobin is considerably more stable, and its unfolding is a two step process (Hughson, F. M., Wright, P. E., and Baldwin, R. L. (1990) Science 249, 1544-1548). After myoglobin loses hemin, the native apomyoglobin denatures to a molten globule intermediate state resulting from unfolding of the B, C, and E helices. The remaining A, G, and H helices unfold during the transition from the intermediate state to the completely unfolded state (Balastrieri, C., Colonna, G., Giovane, A., Irace, G., and Servillo, L., (1976) Methods Enzymol. 76, 72-77; Barrick and Baldwin, 1993; Hughson, F. M., Wright, P. E., and Baldwin, R. L. (1990) Science 249, 1544-1548; Hargrove, M. S., Krzywda, S., Wilkinson, A. J., Dou, Y., Ikeda-Saito, M., & Olson, J. S. Biochemistry 33, 11767-11775, 1994).
Although myoglobin is a useful model system for the alpha and beta subunits of hemoglobin, the effects of mutagenesis of key residues in myoglobin do not always have the same effects when introduced into the hemoglobin subunits. In fact, much has been learned from the differences. This point is best illustrated by the ligand binding studies of genetically engineered His(E7) and Val(E11) mutants of myoglobin, alpha subunits, and beta subunits (Carver, T. W., Rohlfs, R. J., Olson, J. S., Gibson, Q. H., Blackmore, R. S., Springer, B. A. and Sligar, S. G., J. Biol. Chem., 265: 20007-20020; Matthews, A. J., Rohlfs, R. J., Olson, J. S., Tame, J., Renaud, J., & Nagai, K. J. Biol. Chem. 264, 16573-16583, 1989; Mathews, A. J., Olson, J. S., Renaud, J. -P., Tame, J., & Nagai, K. (1991) J. Biol. Chem. 266, 21631-21639; Springer, B. A., Sligar, S. G., Olson, J. S., and Phillips, G. N., Jr. Chem. Rev. 94, 699-714, 1994). From a comparison of the observed effects on oxygen and carbon monoxide rate constants, a general rule has emerged. Myoglobin and R-state hemoglobin alpha subunits seem to have similar distal pocket structural and chemical mechanisms that discriminate against CO in favor of O.sub.2. On the other hand, R-state beta subunits appear to have evolved somewhat different distal pocket mechanisms that accomplish the same physiological functions. In addition, hemoglobin interconverts between the R- and T-states. The alpha and beta subunits may have different structural and chemical mechanisms in each conformation. These observations raise the question as to whether or not mutations that have favorable effects on myoglobin stability will have similar effects in the hemoglobin subunits.